The gene regulatory networks active in early development set up tissue-specific patterns of gene expression, but little is understood about how the restricted expression of large sets of tissue-specific effector genes leads to the unique aspects of morphogenesis and differentiation in different tissues. These effector gene sets have complex regulatory and functional interactions that have proven very challenging to dissect. Despite this difficulty, understanding morphogenetic effector networks is fundamental to developmental biology and is integral to regenerative medicine, tissue engineering and the understanding and treatment of birth defects. The proposed research integrates state of the art methods for transcriptional profiling, targeted gene disruption, and quantitative multidimensional imaging into a systems biology approach to dissecting morphogenetic effector networks in a carefully chosen model organ, the Ciona notochord. Ascidians such as Ciona are close chordate relatives of the vertebrates with extensively conserved embryonic anatomy, but with a particularly small, simple embryo, a very compact genome and unusually straightforward transgenesis. The Ciona notochord undergoes a broad range of complex, conserved, medically relevant morphogenetic behaviors but consists of only 40 cells that can easily be imaged in their entirety with fine subcellular detail. Aim 1 is to identify all the genes expressed and upregulated in the notochord using a direct RNA sequencing approach on purified notochord and not-notochord cells from timepoints spanning key steps in morphogenesis. The overarching hypothesis is that genes specifically expressed in the notochord will prove to have distinct roles in notochord morphogenesis and differentiation, and that identifying the full suite of these genes will be essential for building comprehensive models of regulatory and functional interactions. Aim 2 is to systematically disrupt a large and carefully chosen subset of these genes and quantify the resulting phenotypes across many dimensions of cell size, shape and tissue architecture. This will enable the identification of both major and minor players in notochord morphogenesis and will allow these genes to be linked into networks of phenotypic similarity with important functional implications. Aim 3 is to use RNA sequencing on embryos in which each of the transcription factors upregulated in the notochord has been disrupted so as to identify transcriptional regulatory relationships genome-wide. Chromatin immunoprecipitation sequencing will be used to test if these relationships are direct or indirect and thus to build a comprehensive effector gene regulatory network for the notochord. Together these aims will allow a systematic dissection of the transcriptional regulatory architecture of morphogenesis and differentiation in this tractable model organ with unprecedented scope and detail. This new ability to relate gene regulatory network structure to effector gene function will be a major step towards an integrative understanding of how genome sequences encode the dynamic cell properties of differentiating tissues.